Elastin protective polyphenolics and methods of using the same

ABSTRACT

Dermal fibroblasts permanently loose their ability to synthesize elastin, the major component of elastic fibers, shortly after puberty. This progressive loss of elastic fibers cannot be replaced, resulting in the physical signs of aging. The present invention provides methods and compositions containing the polyphenols ellagic acid and/or tannic acid for protection against degradation of cutaneous elastic fibers by the elastolytic enzymes. The use of ellagic acid and/or tannic acid increased the overall deposition of elastic fibers in healthy and damaged skin cells. The protection of both intra-tropoelastin and extra-cellular mature elastic fibers from proteolytic enzymes by ellagic acid and tannic acid caused an increase in the net deposition of elastic fibers. Therefore, embodiments of the present invention provide methods and composition for the treatment of skin and prevention and treatment of degradation of dermal elastic fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/924,586 filed Oct. 25, 2007, which is a continuation-in-part of U.S.patent application Ser. No. 11/394,354 filed Mar. 29, 2006, now U.S.Pat. No. 7,566,693, issued Jul. 28, 2009; which claims domestic priorityfrom U.S. Provisional Application No. 60/758,821 filed Jan. 13, 2006;U.S. Provisional Patent Application No. 60/737,586 filed Nov. 17, 2005;U.S. Provisional Patent Application No. 60/681,600 filed May 17, 2005;U.S. Provisional Patent Application No. 60/671,557 filed Apr. 15, 2005;and U.S. Provisional Application No. 60/665,966 filed Mar. 29, 2005, thedisclosures of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH PROJECTS

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NAMES OF PARTIES SUBJECT TO A JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A DISC

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BACKGROUND

Children with inherited diseases, characterized by impaired primarydeposition of elastic fibers (i.e. Costello Syndrome or Cutis Laxa)develop wrinkles and deep dermal creases. Similar, but steadilydeveloping signs of premature skin aging can also be observed inindividuals with Pseudoxanthoma Elasticum and in normal persons afterprolonged exposure to sun. Histological analysis of wrinkled skindemonstrates disappearance and altered organization of elastic fibersdue to premature proteolytic degradation and impaired remodeling (solarelastosis) of these components of dermal extracellular matrix. Thisobserved loss of physiologically relevant elastic fibers is alsoaffected by the fact that fully differentiated (adult) dermalfibroblasts lose their ability to synthesize elastin and thus cannotreplace damaged elastic fibers. Since elastic fibers are solelyresponsible for cutaneous elasticity/resilience there is an obvious needfor development of methods that might protect existing elastic fibersfrom premature degradation by elastolytic proteinases and facilitate newelastogenesis in skin.

A proteolytic digest of elastin to provide a mixture of smallelastin-derived peptides (ProK-60), manganese salts (MnCl₂, MnSO₄ andMnPCA) and trivalent iron (Ferric Ammonium Citrate) have each been shownto individually stimulate the production and effective assembly of newtropoelastin into new elastic fibers in both primary cultures of humandermal fibroblasts and in organ cultures of human adult skin explants.Yet even under optimal conditions, the elastogenic process is not 100%efficient. A significant fraction (30-40%) of newly producedtropoelastin is not assembled into extracellular elastic fibers.Instead, these unassembled tropoelastin interact with the cell surfaceelastin receptor and further stimulate new elastogenesis, pro-mitogenicsignaling pathways and pro-migratory signaling pathways. Moreover, theseunassembled tropoelastin molecules and the soluble products ofproteolytic degradation of insoluble elastin can stimulate the secretionof elastolytic metalloproteinases. While stimulation of dermalfibroblast proliferation and migration can contribute to the overallanti-aging effect induced by factors initially triggering newelastogenesis, the simultaneous up-regulation of elastolytic enzymes maycause rapid degradation of newly produced elastin and existing elasticfibers. Hence there is a need to protect existing and new elastic fibersfrom premature enzymatic proteolysis.

It has now been shown that the treatment of cultured dermal fibroblastswith ellagic acid or tannic acid significantly enhances their netdeposition of elastic fibers. This effect is due to the fact that thesereagents bind to the newly produced elastin and protect it fromproteolytic degradation.

Ellagic acid and tannic acid are polyphenols found in a wide variety offruits and nuts such as raspberries, strawberries, walnuts, grapes, andblack currants. These molecules possess potent ability to scavengereactive oxygen species (ROS) and reactive nitrogen species (RNS). BothROS and RNS, generated inside cells after exposure to several endogenousand exogenous agents, may cause direct or indirect damage of manyimportant biomolecules, including elastin mRNA, by activation of localproteinases, glycosidases or RNAses. Moreover, tannic acid has beenshown to bind to insoluble bovine and porcine elastin and inhibit theirdegradation by porcine pancreatic elastase and recently, ellagic acidwas shown to decrease expression of pro-MMP-2 and pro-MMP-9, precursorsof two elastolytic enzymes.

The most extensively studied polyphenol, ellagic acid, exhibits minimalsolubility in water and moderate to better solubility in organicsolvents such as methanol and DMSO, suggesting that ellagic acid may actas a good lipophilic antioxidant. Experimental data indicate thatellagic acid inhibits lipid peroxidation at much lower concentrationsthan vitamin E. This property, along with its ability to scavengeperoxyl radicals, makes it a probable chain-breaking antioxidantcandidate.

Epidemiological studies indicate that there is an inverse associationbetween the incidence of coronary heart diseases and fruit consumption,largely attributed to the antioxidant nature of phenolic compounds.Ellagic acid exhibits cardio-protective properties in the neoepinephrinemyocarditis rat model, hepato-protective activity against carbontetrachloride both in vitro and in vivo and reduced cytogenetic damageinduced by radiation, hydrogen peroxide and mitomycin C.

Additional experimental studies have demonstrated that ellagic acid,tannic acid and their derivatives, due to their planar structure, alsobind to DNA by intercalating into the minor groove and exhibitanti-mutagenic, anti-cancer and anti-proliferative activities. Inaddition, ellagic acid induces G1 arrest and inhibits overall cellgrowth, causing apoptosis in several tumor cells. Ellagic acid has alsobeen shown to inhibit chemically induced cancer in the lung, liver, skinand esophagus of rodents, including TPA-induced tumor promotion in mouseskin. Given the common etiopathogenic processes of mutagenesis,carcinogenesis, and teratogenesis induced by genotoxic chemicals,ellagic acid was also tested for embryoprotection and demonstrated thatit can interrupt the critical teratogenic events induced by methylatingagents.

Topical applications of ellagic acid have been used in therapeuticpreparations. Gali, et. al. demonstrated that topical applications oftannic acid practically inhibit tumor promoter-induced ornithinedecarboxylase activity (ODA) in mouse epidermis in vivo suggesting thattannic acid and other polyphenols may be effective not only against skintumor initiation and complete carcinogenesis, but also against thepromotion phase of skin tumorigenesis. Moreover, tannic acid and itspolyphenol derivatives have been shown to possess anti-inflammatoryactivities and to decrease infectivity of human cells with papilomavirus, human immunodeficiency virus, and Staphylococcus aureus.

BRIEF SUMMARY OF THE INVENTION

The present invention provides evidence that the effectiveness ofellagic acid or tannic acid to prevent premature proteolytic degradationof tropoelastin and fully polymerized elastin, thus facilitating moreefficient elastogenesis. Thus, embodiments of the present inventionprovide compositions and methods for treating aging or damaged skinusing ellagic acid, tannic acid, or derivatives thereof.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of necessary fee.

FIG. 1. Assessment of immunodetected and insoluble elastic fibers indermal fibroblast cultures. (a) Micrographs of immunodetectedtropoelastin in cultures maintained in the presence and absence of EAand TA (1 μg/mL each). (b) Results of morphometric evaluation oftropoelastin levels in fibroblast cultures. (c) Evaluation ofmetabolically labeled insoluble elastin in treated and control cultures.All results were obtained in 7 day-old cultures of dermal fibroblastsderived from a 36 year-old caucasian female. Data demonstrate thattreatment with ellagic acid or tannic acid significantly increases a netdeposition of extracellular elastic fibers as compared to respectiveuntreated controls.

FIG. 2. Pulse and chase experiment to evaluate pretreatedtropoelastin/elastin stability against non-specific enzymaticdegradation. (a) Results of morphometric assessment of immuno-detectableelastin and (b) content of metabolically labeled insoluble elastin,detected at the respective ends of the indicated pulse and chaseperiods, demonstrate that cultures of dermal fibroblasts derived from a26 year-old female, that were incubated the first seven days in thepresence of EA and TA (1 μg/mL each), sustain their high net content ofinsoluble elastin (metabolically pulsed with [³H]-valine between day 4and 7) even when maintained for an additional seven days (chase period)in media containing only 1% FBS and no polyphenols. In contrast, 14day-old control (untreated) cultures demonstrate a significant decreasein their net content of metabolically labeled insoluble elastin(initially deposited at the end of pulse period, at day 7). (c) Theassessment of [³H]-thymidine incorporation and assay of total DNAindicate that conditions of the chase period in which cells weremaintained in 1% FBS caused inhibition of cellular proliferation.

FIG. 3. Evaluation of the protective effect of polyphenols againstelastolytic degradation of insoluble elastin. Results of in vitro assaydemonstrate that samples of insoluble [³H]-labeled elastin from bovineligamentum nuchae, pretreated with EA or TA (1 μg/mL and 10 μg/mL each)demonstrate higher resistance to proteolytic degradation by indicatedenzymes belonging to three different classes of proteinases (elastases)capable of elastin degradation.

FIG. 4. Assessment of the effect of polyphenols on elastogenesis inducedby known elastogenic compounds. (a) Results of the quantitativeassessment of newly deposited insoluble elastin (metabolically labeledwith [³H]-valine) detected in 7 day-old cultures of dermal fibroblastsderived from a healthy 50 year-old caucasian female. Fibroblastsmaintained in the presence of stimulators of elastin synthesis, mixtureof small elastin-derived peptides (ProK-60 25 μg/mL) or Ferric AmmoniumCitrate (FAC 20 μM), significantly increased their net deposition ofinsoluble elastin as compared with the untreated control. Additionaltreatment either with ellagic acid (1 μg/mL) or tannic acid (1 μg/mL)cause further proportional increase in net elastin content in all testedexperimental groups. (b) Representative micrographs of 7 day-oldcultures of dermal fibroblasts (derived from 50 year-old caucasianfemale) immuno-stained with anti tropoelastin antibody. Cultures treatedwith ProK-60 (25 μg/mL) produced more elastic fibers than untreatedcontrol cultures. Additional treatment either with ellagic ecid (EA) orwith tannic acid (TA) (both in concentration 1 μg/mL) caused a furtherincrease in net deposition of immunodetectable elastic fibers.

FIG. 5. Assessment of binding of tannic acid to collagen type I. (a)Results of triplicate (1 mg) aliquots of pure collage type I incubatedwith 20 μg/ml of tannic acid for 2 hour at 37 C. Initial concentrationof tannic acid was confirmed by direct spectrophotometric reading at 280nm. This method demonstrated a dose-dependent linear increase inabsorbance. At the end of incubation period the collagen type I slurrieswere separated by centrifugation and the concentration of TA insupernatants were spectrophotometrically determined again at 280 nm. Ineach experimental group means±SD were calculated and obtained valueswere statistically compared with beginning concentrations of bothpolyphenols. (b) 1 mg of collagen type I (from rat tail) sequestered75.5±0.001% (P<0.0001) of the TA (originally 20 μg/mL) from solution,suggesting that tannic acid may also bind to collagen type I.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%. Generally speaking, the term“tissue” refers to any aggregation of similarly specialized cells whichare united in the performance of a particular function.

The term “cosmetic,” as used herein, refers to a beautifying substanceor preparation which preserves, restores, bestows, simulates, orenhances the appearance of bodily beauty or appears to enhance thebeauty or youthfulness, specifically as it relates to the appearance oftissue or skin.

The term “modify” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the tissue to which it is being provided, applied oradministered. The change in form may be demonstrated by any of thefollowing alone or in combination: enhanced appearance of the skin;increased softness of the skin; increased turgor of the skin; increasedtexture of the skin; increased elasticity of the skin; decreased wrinkleformation and increased endogenous elastin production in the skin,increased firmness and resiliency of the skin.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.In a preferred embodiment, the therapeutic composition is notimmunogenic when administered to a human patient for therapeuticpurposes.

“Providing” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “providing”, when used in conjunction with a polyphenolic compound,can include, but is not limited to, providing an polyphenolic compoundinto or onto the target tissue; providing a polyphenolic compoundsystemically to a patient by, e.g., intravenous injection whereby thetherapeutic reaches the target tissue; and the like.

The term “skin” means that outer integument or covering of the body,consisting of the dermis and the epidermis and resting upon subcutaneoustissue.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto improve the functionality, the appearance, the elasticity, and/or theelastin content of mammalian tissue. As it applies to skin, it ismeasured by turgor, tone, appearance, degree of wrinkles, andyouthfulness. As the term applies to blood vessels it may be measured bythe degree of elasticity or proper vasomotor response(vasodilatation/vasoconstriction) of the vessel. Accordingly,therapeutic treatment of blood vessels may have implications in diseasesassociated with visco-elasticity, including hypertension,arteriosclerosis, angina, angiogenesis, myocardial infarction, coronarythrombosis, restenosis post angioplasty, and chronic obstructivepulmonary disease.

The terms “therapeutically effective” or “effective”, as used herein,may be used interchangeably and refer to an amount of a therapeuticcomposition of the present invention—e.g., a polyphenolic compound. Forexample, a therapeutically effective amount of a composition comprisingpolyphenolic compound is a predetermined amount calculated to achievethe desired effect, i.e., to effectively promote elastin production,cell proliferation, or improved appearance, or improved tissueelasticity in an individual to whom the composition is administered.

As used herein, “tissue”, unless otherwise indicated, refers to tissuewhich includes Elastin as part of its necessary structure and/orfunction. For example, connective tissue which is made up of, amongother things, collagen fibrils and elastin fibrils satisfies thedefinition of “tissue” as used herein. Additionally, elastin appears tobe involved in the proper function of blood vessels, veins, and arteriesin their inherent visco-elasticity.

Skin is composed of a top layer, the epidermis, which is approximately20 cell layers or about 0.1 mm in thickness, and a lower layer, thedermis, which is from about 1 to about 4 mm in thickness and containssmall blood vessels, collagen, elastin and fibroblasts. The dermisprovides structural support and nutrients to the epidermis. Aging hasbeen shown to increase cellular heterogeneity of the epidermal layer,however, it has little effect on the thickness of the epidermal layer.The supporting dermis, on the other hand, is known to thin with age andexposure to the sun and environmental contaminants (other environmentaleffects on the skin are discussed in U.S. Pat. Nos. 4,938,969 and5,140,043, the disclosure of which is herein incorporated by reference).As the dermal layer provides the support and blood supply for theepidermis, the dermal layer is important in maintaining the elasticityand appearance of the skin. Disruption of the supporting dermis leadsdirectly to sagging and, consequently, furrowing of the epidermis, i.e.,the formation of wrinkles.

Deep wrinkles are also due to continual stretching and contraction ofboth the dermis and epidermis. Currently, these deep wrinkles or furrowsmay only be eliminated by plastic surgery or by collagen injectionsdirectly beneath the depressed areas. The fine wrinkles that occur withage and prolonged exposure to the sun and other environmentalcontaminants are the direct result of deterioration of the supportingdermal layer.

Elastin is secreted by the fibroblasts of the connective tissues and bythe vascular smooth muscle cells (i.e., arteries, veins and heart) andelastic cartilage chondrocytes (i.e., epiglottis and ear cartilage) intothe extracellular matrix. In the dermal connective tissue, the elastinfibers are thin and sinuous. Elastin contained in the dermis represents5% of its dry weight. Elastin is a large fibrous protein which is formedby spiral filaments that can be compared to springs. The spiralfilaments consist of peptidic chains that can stretch out. The peptidicchains are connected to each other by very specific amino-acids:desmosin and isodesmosin, which builds between them, giving the moleculea reticular aspect. After stretching out, the molecules resume theiroriginal shape due to this cross linking, which is essential tomolecular elasticity.

The biosynthesis of elastin begins with the embryonic period andcontinues through adulthood, at which time our body stops producingelastin. Thus, elastin is no longer renewed. With aging, the elasticfibers progressively degenerate and separate into fragments. The skinprogressively loses its elasticity, resulting in fine lines andwrinkles. This damage to our elastic tissue cannot be avoided and ispart of the natural (physiological) aging process. This process beginsrelatively early, but accelerates considerably after age 40.

Elastin owes its properties to its thin structure which resembles thatof rubber. Elastin is the protein responsible for our skin's essentialelasticity and tonicity. Its decrease means the skin starts sagging,allowing fine lines, folds and wrinkles to appear and grow.

One embodiment of the present invention provides compositions comprisingat least one polyphenolic compound, or derivatives thereof, preferablyellagic acid or tannic acid or a combination thereof. The polyphenoliccompound may be present in an effective amount, for example, tostimulate elastogenesis or protect elastin fibers from degradation. Inone embodiment, an effective amount is from about 0.01 μg to about 100μg, preferably from about 1 μg to about 10 μg.

Compositions of the present invention may further include a stimulatorof elastogenesis. Such stimulators of elastogenensis include, but arenot limited to, elastin derived peptides, plant derived peptides, bovinederived peptides, manganese, iron, copper and combinations thereof.

Embodiments of the present invention may further comprise an agentselected from anti-inflammatory agents, sunscreens, sunblocks,stimulators of protein synthesis, cell membrane stabilizing agents,moisturizing agents, coloring agents, opacifying agents and combinationsthereof.

A further embodiment of the present invention provides compositionscomprising at least one polyphenolic compound, or derivatives thereof,preferably ellagic acid or tannic acid and optionally one stimulator ofelastogenesis. A stimulator of elastogenesis may be, for example, smallelastin-derived peptides including, but not limited to, ProK-60 or othersmall elastin-derived peptides as set forth in co-pending U.S.application Ser. No. 10/778,253 entitled “Elastin Digest Compositionsand Methods Utilizing the Same” filed Feb. 13, 2004, the contents ofwhich are herein incorporated by reference in its entirety. Suchcompositions may be useful to significantly increase the net depositionof insoluble elastic fibers, thereby enhancing the skin's elasticity anddecreasing the appearance of fine lines and/or wrinkles. Thus, furtherembodiments of the present invention provide compositions and methods tocompensate for the loss of elastic components in the dermis.

The result of aging on skin, whether or not it has been accelerated byenvironmental damage (such as radiation, pollution) is a deteriorationof the dermal layer—fewer fibroblasts, less collagen, less elastin andless circulatory support. Consequently, the normal stretching andcontraction of the skin leads to damage of the dermis that is notreadily corrected, resulting in wrinkling. Further embodiments of thepresent invention provide methods and compositions for increasing thedeposition of insoluble elastin fibers, therein reducing the effects ofradiation, including, but not limited to, ultraviolet radiation, orother environmental damage.

Dermatologists and cosmetologists have directed their efforts toimproving the appearance of skin using agents known to stimulate thegrowth and proliferation of epidermal cells. Newly proliferated cellsprovide more structure and hold more moisture, giving the skin a youngerappearance. One method of causing new skin cell proliferation isaccomplished by use of an irritant or chemical peel in which theuppermost layers of the epidermis are caused to slough off, leading toproliferation and replacement with new epidermal cells. While suchtreatment is recognized to provide some cosmetic improvement, it doesnot address the major causative factor—the compromised supporting dermallayer. Thus, embodiments of the present invention also provide methodsand compositions for the enhanced deposition of insoluble elastinfibers, therein providing the dermal support and elasticity necessaryfor smooth, supple skin.

One embodiment of the invention is a stable, effective topicalcomposition comprising at least one polyphenolic compound, or derivativethereof. Preferably, the polyphenolic compound is selected from tannicacid, ellagic acid, derivatives thereof, salts thereof and combinationsthereof.

Another embodiment of the present invention is a method of treatingdamage to skin, such as often arises due ultraviolet light exposureand/or aging. The method includes applying the present topicalcomposition to a damaged portion of the skin, for example, but notlimited to, topically applying compositions of the present invention tothe locus of wrinkles. These topical polyphenolic based compositions areparticularly effective for reducing epidermal wrinkling resulting fromintrinsic aging, photo damage, or other environmental damage.

Such compositions may also be used prophylactically to reducephoto-induced damage which can result from exposure of skin to sunlightand other harmful irradiation.

Another embodiment of the invention is a method of prophylacticallyapplying the compositions of the invention for the protection of theskin against damage which may occur due to radiation or otherenvironmental insults/exposure.

A further embodiment provides a method for treating and/or reducingwrinkles and/or fine lines by contacting skin with a composition of thepresent invention.

Another embodiment of the invention is a method for stabilizinginsoluble elastin fibers present in the skin by contacting the skin witha topical composition of the invention. An object of the presentinvention is to provide a composition useful in minimizing early andacute ultraviolet radiation damage, as well as late and chronicradiation induced photo damage which together may enhance or causephotoaging of the skin.

It is yet a further object of the present invention to provide in theform of a topical carrier, at least one polyphenolic compound, orderivative thereof, including, but not limited to, tannic acid and/orellagic acid, which is effective in increasing the amount of insolubleelastin deposition, therein restoring that elastin which is degradedupon exposure to free radicals.

It is another object of the present invention to provide factors,including but not limited to, a source of small elastin-derived peptides(for example, ProK-60), a manganese component (for example, Mn-PCA,manganese sulfate, manganese gluconate), an iron component (for example,ferric ammonium citrate), a copper component (for example, copper-PCA,copper sulfate), bovine-derived peptides or plant-derived peptides topromote elastogenesis for skin repair and wound healing that occurs dueto photoaging processes in the skin, such as those that can occur fromacute sunburn and/or chronic exposure to ultraviolet radiation. Suitablemanganese and iron components are described in co-pending U.S.application Ser. No. 11/062,377 entitled “Compositions for Elastogenesisand Connective Tissue Treatment” filed Feb. 22, 2005, suitableplant-derived peptides are described in co-pending U.S. Application No.60/671,557 entitled “Plant-Derived Elastin Binding Protein Ligands andMethods of Using the Same” filed Apr. 15, 2005, and suitablebovine-derived peptides are described in co-pending U.S. Application No.60/681,600 entitled “Proteolytic Digest Derived from Bovine LigamentumNuchae Stimulates Deposition of New Elastin-Enriched Matrix in Culturesand Transplants of Human Dermal Fibroblasts” filed May 17, 2005, thecontents of which are all herein incorporated by reference in theirentireties.

A further embodiment of the present invention provides a compositioncomprising an effective amount of a polyphenolic compound and an elastinderived peptide is provided. The polyphenolic compound may be selectedfrom tannic acid, ellagic acid and combinations thereof. An effectiveamount of the polyphenolic compound is preferably from about 1 μg toabout 10 μg. In a preferred embodiment, the elastin derived peptide isselected from ProK60, E91 and a combination thereof.

In a further embodiment of the present invention a compositioncomprising an effective amount of a polyphenolic compound and anelastogenic plant-derived peptide is provided. The polyphenolic compoundmay be selected from tannic acid, ellagic acid and a combinationthereof. An effective amount of the polyphenolic compound is preferablyfrom about 1 μg to about 10 μg.

Another embodiment is a method for protecting elastin fibers fromdegradation comprising administering an effective amount of apolyphenolic compound to a subject in need thereof. The polyphenoliccompound is selected from tannic acid, ellagic acid and a combinationthereof. An effective amount of the polyphenolic compound is preferablyfrom about 1 μg to about 10 μg.

In another embodiment, the method may further comprise administering astimulator of elastogenesis selected from elastin-derived peptides,plant-derived peptides, bovine-derived peptides, manganese, iron, copperand combinations thereof. The polyphenolic compound and the stimulatorof elastogenesis may be administered simultaneously or sequentially.

The preparation of a pharmacological composition that contains activeingredients dispersed therein is well understood in the art. Typicallysuch compositions if desired, may be prepared as sterile compositionseither as liquid solutions or suspensions, aqueous or non-aqueous,however, suspensions in liquid prior to use can also be prepared.

The active ingredient of the present invention may be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Various excipients may be used as carriers forthe peptide compositions of the present invention as would be known tothose skilled in the art. For example, compounds may be dissolvedexcipients such as water comprising solutions, alcohol comprisingmixtures, intravenous and saline comprising mixture, dextrose, glycerol,ethanol or the like and combinations thereof. In addition, if desired,the composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient.

Formulations comprising polyphenolics, for example ellagic acid ortannic acid, may be prepared by mixing such excipients with thepolyphenolic. The polyphenolic compounds in the formulation may comprisefrom about 0.0002 to about 90% by weight of the formulation. Theseformulations may be employed directly as a constituent of therapeutic orcosmetic treatments, such as emulsions, lotions, sprays, ointments,creams and foam masks. Final products may contain up to 10% by weightbut preferably 0.001 to 5% of such a solution though of course moreconcentrated or more dilute solutions may also be used in greater orlesser amounts. For example, an eye cream may comprise about 0.1% (w/w)and a facial cream may comprise about 0.01% (w/w) of a polyphenoliccompound in an excipient.

A therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts thatare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, tartaric, mandelicand the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

Physiologically tolerable carriers and excipients are well known in theart. Other equivalent terms include physiologically acceptable or tissuecompatible. Exemplary of liquid carriers are sterile aqueous solutionsthat contain no materials in addition to the active ingredients andwater, or contain a buffer such as sodium phosphate at physiological pHvalue, physiological saline or both, such as phosphate-buffered saline.Still further, aqueous carriers can contain more than one buffer salt,as well as salts such as sodium and potassium chlorides, dextrose,propylene glycol, polyethylene glycol and other solutes.

Thus, the dosage ranges for the administration of polyphenolic are thoselarge enough to produce the desired effect in which the condition to betreated is ameliorated. The dosage should not be so large as to causeadverse side effects. Generally, the dosage will vary with the age,condition, and sex of the patient, and the extent of the disease in thepatient, and can be determined by one of skill in the art. The dosagecan be adjusted in the event of any complication.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. A therapeuticamount of a polyphenolic-based composition is an amount sufficient toproduce the desired result, and can vary widely depending upon thedisease condition and the potency of the therapeutic compound. In thepresent invention the desired result is an improvement in elasticity ofthe tissue as determined by an improvement in the elastin content of thetissue, improved capacity and function of the tissue, or improvedappearance, suppleness, and/or tone of the tissue being treated. Thequantity to be administered depends on the subject to be treated, thecapacity of the subject's system to utilize the active ingredient, andthe degree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the conditions of administration. Suitable regimes for administrationare also variable, but are typified by an initial administrationfollowed by repeated doses at one or more time intervals by a subsequentadministration. Where a single composition is not available for atreatment, or where such a composition is not desirable, administrationof composition may also comprise the application of several differentcompositions sequentially to achieve a desired therapeutic effect.

Topical carriers are employed which should be both non-irritating to theskin and which are suitable for delivering the active components to theskin. Further, suitable topical carriers should be those which do notinhibit the antioxidant activity of the active ingredients thus reducingthe efficiency of the composition for protecting the skin from theeffects of acute and chronic ultraviolet radiation. Further, suchcarriers must be of sufficiently high purity and sufficiently lowtoxicity to render them suitable for chronic topical administration tothe skin and be free of bacterial contaminants.

The active ingredients described herein can be incorporated in anysuitable pharmacologically acceptable carrier which is suitable fortopical administration to the human skin. As such, the pharmacologicallyacceptable carrier must be of sufficient purity and have sufficientlylow toxicity to render it suitable for administration to a human notingthat, generally, the carrier can represent up to 99.99% and typicallyfrom at least approximately 80% of the total composition. Thus, thephrase “pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The pharmaceutically acceptablecarriers and additives employed in the present compositions arecompatible with at the tannic acid and/or ellagic acid compounds andcompositions described herein containing such compounds.

Typical compositions for use herein include a wide variety of physicalforms. These include, but are not limited to, solutions, lotions,creams, oils, gels, sticks, sprays, ointments, balms, patches andpastes. Generally, such carrier systems can be described as beingsolutions, creams, emulsions, gels, solids and aerosols.

Solvents are generally employed in the preparation of suitable topicalcompositions. Such solvents can either be aqueous or organic based and,in either case, the solvent must be capable of having dispersed ordissolved therein the above-described active components while not beingirritating to the user. Water is a typical aqueous solvent whilesuitable organic solvents include propylene glycol, battalion glycol,polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol,sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol andmixtures thereof. Solvents can be included in the overall composition inamounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. It isnoted that compositions of the present invention can be produced in theform of an emollient. A wide variety of suitable emollients are knownand may be used herein. In this regard, reference is made to U.S. Pat.No. 5,296,500, the disclosure of which is incorporated by reference.

Alternatively, the polyphenolic compound can be formulated as a lotioncontaining from about 0.01% to 10% of the above described activeingredients. Further, it may be formulated from a solution carriersystem as a cream. A cream of the present invention would preferablycomprise from about 0.1% to 15% and preferably from 1% to 5% of theabove described active ingredients. Lotions and creams can be formulatedas emulsions as well as solutions.

It is contemplated that the polyphenolic compounds described above maybe used as a lotion or cream emulsion of the oil-in-water type or as awater-in-oil type, both of which being extremely well known in thecosmetic field. Multi-phase emulsions such as the water-in-oil type isdisclosed in U.S. Pat. No. 4,254,105, the disclosure of which isincorporated herein by reference, may also be employed.

It is further contemplated that the polyphenolic compounds be formulatedfrom a solution carrier system as an ointment. An ointment may comprisea simple base of animal or vegetable oils or semi-solid hydrocarbons(oleaginous). Ointments may also comprise absorption ointment baseswhich absorb water to form emulsions. Ointment carriers may also bewater soluble. An ointment may comprise from 1% to 99% of an emollientplus to about 0.1% to 99% of a thickening agent. Reference is again madeto U.S. Pat. No. 5,296,500 and the citations contained therein for amore complete disclosure of the various ointment, cream and lotionformulations for use herein.

The compositions can include one or more of a variety of optionalingredients, such as, but not limited to, anti-inflammatory agents,sunscreens/sunblocks, stimulators of protein synthesis, cell membranestabilizing agents (i.e., carnitine), moisturizing agents, coloringagents, opacifying agents and the like, so long as they do not interferewith the elastin stabilizing properties of the polyphenolic compounds,or derivatives thereof. The formulation can also include, other activeingredients, such as antibiotics, analgesics, anti-allergenics and thelike. The formulation is commonly applied to the skin as a lotion orcream to be rubbed on body tissue over the desired area. For optimumefficacy treatment in accordance with the presented method should beinitiated as early as possible following exposure to sunlight or otherradiation source. The formulation is generally applied to the skin onceor twice daily. As noted elsewhere herein, the present composition mayalso be used to inhibit and/or minimize the effects of aging and/orphoto damage on the skin.

In the compositions provided herein the polyphenolic compounds, orderivatives thereof, such as tannic acid and/or ellagic acid, is presentin an amount from about 0.01 to 80 weight percent, further from about0.1 to 20 weight percent, and further from about 0.5 to 10 weightpercent.

The optional source of small elastin-derived peptides (such as, but notlimited to ProK-60 or E91) component stimulates new elastogenesis,supplementing elastic tissue, and consequently, reduction of wrinklesand other skin conditions related to loss of elasticity. The source ofsmall elastin-derived peptides component, when used in the compositionis generally present in an amount from about 0.001 to about 10 weightpercent, preferably from about 0.005 to about 0.1 weight percent of thecomposition.

The optional manganese component may be any magnesium compound, or apharmaceutically acceptable salt thereof, but preferably is MnCl₁, MnSO₄and/or MnPCA, wherein the manganese component is typically present in anamount from about 0.001 to 10 weight percent, preferably from about0.0012 to 0.012 weight percent of the composition.

A trivalent iron component, such as, but not limited to, Ferric AmmoniumCitrate (FAC) may also be included in the composition. The trivalentiron component stimulates new elastogenesis and assists in treatment ofelastic tissue defects. The trivalent iron, when included in thecomposition, is generally present in an amount from about 0.001 to about10 weight percent of the composition.

Copper may also be included in the composition. In preferredembodiments, copper may be present in about 0.001 to about 10 weightpercent, more preferably from about 0.005 to about 0.1 weight percent ofthe composition.

Elastogenic plant-derived peptides may also be present in thecomposition. Such peptides are more fully described in U.S. ProvisionalPatent Application No. 60/671,557 filed Apr. 15, 2005 entitled“Plant-Derived Elastin Binding Protein Ligands and Methods of Using theSame”, U.S. Provisional Application No. 60/681,600 filed May 17, 2005entitled “Proteolytic Digest Derived from Bovine Ligamentum NuchaeStimulates Deposition of New Elastin-Enriched Matrix in Cultures andTransplants of Human Dermal Fibroblasts” and U.S. Provisional PatentApplication No. 60/737,586 filed Nov. 17, 2005 entitled “Plant-DerivedElastin Binding Protein Ligands and Methods of Using the Same. Suchpeptides may be sextapeptide comprising the sequence X1-X2-X3-X4-X5-X 6(SEQ ID NO 18), wherein X1 is V or I, X2 is G, X3 is A, L or V, X4 is M,S, or A, X5 is P and X6 is G. Such a plant-derived peptide or syntheticplant-derived peptide may be present in the composition. In preferredembodiments, the peptide may be present in about 0.0001 to about 0.01weight percent, more preferably from about 0.0004 to about 0.002 weightpercent of the composition.

Upon formulation, compositions of the present invention may beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective. The formulations are easilyadministered in a variety of dosage forms such as direct topicalapplication, application via a transdermal patch and the like.

For topical administration in an aqueous solution, for example, thecompositions may be used directly on the skin without any toxic effectsto the patient. Alternatively, the compositions of the invention may bedissolved or resuspended in a suitable buffer prior to mixing, ifnecessary.

In general, routine experimentation will determine specific ranges foroptimal therapeutic effect for each composition and each administrativeprotocol, and administration to specific individuals will be adjusted towithin effective and safe ranges depending on the condition andresponsiveness of the individual to initial administrations.

Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by the FDA.

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention. Various modifications and combinations of the illustrativeembodiments, as well as other embodiments of the invention, will beapparent to persons skilled in the art upon reference to thedescription.

EXAMPLE 1

Materials. All chemical-grade reagents were obtained from Sigma (St.Louis, Mo.). αMEM medium, fetal bovine serum (FBS), 0.2% trypsine-0.02%EDTA and other cell culture products were obtained from GIBCO LifeTechnologies (Burlington, Canada). Polyclonal antibody to tropoelastinand BA4 monoclonal antibody to VGVAPG (SEQ ID NO 19) were purchased fromElastin Products Company, Inc. (Owensville, Mo.). Monospecificpolyclonal anti-AKAAAKAAAKA (SEQ ID NO 20) antibody was a gift of Dr.Barry Starcher from the University of Texas. Secondary antibodyfluorescein-conjugated goat anti-rabbit (GAR-FITC) was purchased fromSigma (St. Louis, Mo.). DNeasy Tissue system for DNA assay and RNeasyMini Kit for isolation of total RNA were purchased from Qiagen(Mississauga, Canada). Expression probe for elastin was purchased fromApplied Biosystems (Foster City, Calif.). The radiolabeled reagents,[3H]-valine, and [3H]-thymidine were purchased from Amersham Canada Ltd.(Oakville, Canada).

Cell Cultures. Biological effects of ellagic acid and tannic acid weretested in cultures of dermal fibroblasts derived from punch biopsies ofhealthy skin from Caucasian females of different ages ranging from 4-52years old. All fibroblasts were originally isolated by allowing them tomigrate out of skin explants and then passaged by trypsinization andmaintained in alpha-minimum essential medium supplemented with 20 mMHepes, 1% antibiotics and antimycotics, 1% L-Glutamate and 2% fetalbovine serum (FBS). In all experiments, consecutive passages 3-5 weretested. Cells were densely plated (50×10⁵ cells/dish) to reachconfluency and then cultured for 7 days in the presence and absence ofellagic acid (dissolved in DMSO) and tannic acid (dissolved in water),both in concentration of 1 μg/mL. This optimal concentration was chosenafter a series of pilot experiments indicated that 1 μg/ml of EA and TAinduced optimal effect on net deposition of elastin and did not triggerany change in cellular proliferation rate nor affect basic metabolicperformance.

In a parallel series of experiments, cultures of dermal fibroblasts weretreated with well established stimulators of tropoelastin synthesis,ProK-60 (25 μg/ml) and Ferric Ammonium Citrate (20 μM), andsimultaneously incubated in the presence and absence of ellagic acid ortannic acid (both in concentration 1 μg/ml).

Assessment of Cellular Proliferation. Cellular proliferation rates ofcontrol, ellagic acid and tannic acid treated fibroblasts were assessedat the end point by counting of trypsinized cells, by total DNA assayusing the DNeasy Tissue System from Qiagen and by assessment of[³H]-thymidine incorporation, which was added to all cultures (2μCi/well) for the last 24 hours.

Assessment of Elastin mRNA levels. Fibroblasts were cultured toconfluency in medium with 2% FBS and then in serum-free medium for 24hours. The medium was changed again and cells were incubated for thenext 24 hours in the presence and absence of ellagic acid or tannic acid(both in concentration 1 μg/mL). At the end of the incubation periodtotal RNA was extracted using TRI-reagent. Steady-state levels ofelastin mRNA were analyzed by semi quantitative PCR and by Northern Blotusing a human elastin cDNA recombinant H-11 probe. In all experiments,performed in triplicate, the loading control was routinely performed.

Assessment of elastic fibers content by immunohistochemistry.Seven-day-old and 14-day-old confluent cultures of fibroblasts, whichproduce abundant ECM, were assessed. All cultures were fixed in cold100% methanol at −20° C. for 30 min, then incubated for 1 hour with 2μg/ml of polyclonal antibody to tropoelastin. Cultures were thenincubated for an additional hour with appropriate fluorescein-conjugatedsecondary antibody (GAR-FITC). Nuclei were counterstained with propidiumiodide. Morphometric analysis of five separate cultures in eachexperimental group, immunostained with antibodies recognizingextracellular matrix components was performed using a computerized videoanalysis system (Image-Pro Plus software 3.0, Media Cybernetics, SilverSpring, Md.).

Radioactive metabolic labeling and quantification of newly depositedinsoluble elastin. Quintuplicate, 4 day-old cultures of dermalfibroblasts maintained in the presence or absence of 1 μg/mL of ellagicacid or tannic acid were additionally exposed for the 3 following daysto 20 μCi [³H]-valine. At the end of incubation period the contents ofradioactive NaOH-insoluble elastin was assessed separately in eachculture by scintillation county. Final results reflecting amounts ofmetabolically labeled, insoluble elastin were expressed as CPM/μg DNA.DNA was determined with the DNeasy Tissue System from Qiagen Proteolytic

Pulse and chase experiments aimed at the assessment of durability ofnewly deposited metabolically-labeled elastin. In this series ofexperiments, dermal fibroblasts, plated as described above in media with10% FBS, were maintained in the presence and absence of 1 μg/mL of EA orTA for the first 7 days and pulsed with 20 μCi [³H]-valine between day 4and 7. While quadruplicate 7-day-old cultures from each experimentalgroup were directly processed for the assessment of radioactiveNaOH-insoluble elastin, parallel quadruplicate cultures from eachexperimental group were transferred to media containing only 1% FBS,which did not stimulate proliferation and new elastogenesis, andmaintained for the next seven days (chase period) in the absence ofellagic acid or tannic acid. At day 14 these cultures were terminatedand the net content of the radioactive NaOH-insoluble elastin wasassessed as described above.

Organ cultures of explants derived from surgical biopsies of human skinIn order to further test whether ellagic acid and tannic acid wouldpenetrate into skin tissue and enhance elastogenesis, fragments ofnormal skin (from 30 and 34 year old females) obtained during plasticsurgery procedures were tested in organ culture system. Skin fragmentswere cut into multiple 1 mm² pieces and placed on top of metal gridsimmersed in culture medium containing 5% FBS and maintained for 10 daysin the presence and absence of 1 μg/mL of ellagic acid or tannic acidalone or combined with 25 μg/ml of ProK-60. The media were changed everysecond day. All organ cultures were fixed in 1% buffered formalin andtheir transversal serial histological sections were stained with Movat'spentachrome. Morphometric analysis was performed as described above. Ineach analyzed group (three explants from each patient) low-power fields(1 mm²) of 20 serial sections stained with Movat's pentachrome wereanalyzed and all structures stained black (elastic fibers) were counted.

Assessment of tropoelastin integrity by western blots. To determine theinfluence of tannic acid and ellagic acid on the integrity of solubletropoelastin, dermal fibroblasts obtained from three different donors(initially plated at 50,000 cells/dish) were cultured to confluency inmedium with 5% FBS and then triplicate cultures were incubated for thenext 24 hours in the presence and absence of ellagic acid or tannic acid(both in concentration 1 μg/mL). At the end of the incubation periodconditioned media were collected and then the soluble proteins presentin the intracellular compartments were extracted with 0.5 M acetic acidin the presence of proteinase inhibitors in the following finalconcentrations: 2 mM benzamidine, 2 mM EACA, 2 mM PMSF, 1 mM EDTA and 1mg/ml Trasylol. Extraction was carried out for six hours at 4° C. andthe insoluble material was pelleted by centrifugation. The supernatantwas dialyzed exhaustively (4000 kDa cutoff membrane) at 4° C. againstwater containing proteinase inhibitors, then lyophilized. Concentratedpreparations of the conditioned media and cell extracts from allanalyzed cultures were analyzed for their protein content, and thensamples containing equal amounts of protein (20 μg/sample) weresuspended in 2×SDS sample buffer with DTT, resolved by SDS PAGE,routinely transferred to nitrocellulose and immunoblotted with specificanti-tropoelastin antibody.

Immuno-precipitation of radioactive tropoelastin-like peptides. Thisexperiment was aimed at elucidating whether binding of ellagic acid ortannic acid to tropoelastin molecules would block two characteristicelastin domains responsible for orderly self-aggregation andcross-linking respectively. Instead of very unstable tropoelastin,triplicate samples of a [3H]-valine-labeled recombinant polypeptidecontaining linear amino acid sequences encoded by human tropoelastingene exons; 20-(21-23-24)2 were used. 100 μl samples of this radioactiverecombinant polypeptide modeled after human elastin dissolved in PBS(specific radioactivity 1000 CPM/sample) were incubated in the presenceand absence of 1 μg/mL of ellagic acid or tannic acid, at roomtemperature for 6 hours. Aliquots of all control and experimentalsamples were then immuno-precipitated with (BA4) monoclonal antibody(recognizing VGVAPG (SEQ ID NO 19) and other similar domains, encoded byexons 20 and 24, responsible for self-aggregation of tropoelastin) andwith monospecific polyclonal anti-AKAAAKAAAKA (SEQ ID NO 20) antibodyrecognizing the exon 21- and 23-encoded cross-linking sequences. It wasanticipated that in case that binding of ellagic acid or tannic acidwould block one or both of these crucial domains present in the testedradioactive recombinant peptide, it could not be immunoprecipitated withanti-VGVAPG (SEQ ID NO 19) or anti-AKAAAKAAAKA (SEQ ID NO 20)antibodies.

Proteolytic degradation protection assay of insoluble elastin. Todetermine whether ellagic acid or tannic acid may directly protect fullycross-linked “insoluble elastin” against elastolytic activity of severalelastases, an in vitro assay measuring degradation of an insoluble[³H]-elastin substrate was used. Briefly, insoluble elastin was purifiedfrom bovine ligamentum nuchae using a modification of the hot alkalitechnique and was shown by amino acid analysis to be free ofmicrofibrillar protein and other contaminants. Sequencing of insoluble,digested elastin was performed using an Applied Biosystems model 473Aprotein sequencer equipped with a model 610A data analysis program. Thestock of this pure insoluble elastin preparation was washed twice withwater and twice with acetonitrile and then labeled with sodium[³H]-borohydride and stored at −20° C. Before each experiment, the[³H]-elastin substrate suspended in PBS was boiled for 5 min andextensively washed to remove all unbound radioactivity. Then, its 100 μgaliquots (specific activity 300 CPM/1 μg) were suspended in serum freeculture medium and pre-incubated for 1 hour in the presence and absenceof 1 μg/mL or 10 μg/mL of ellagic acid or tannic acid. All samples ofradioactive elastin were then submitted to three 5 min washes in serumfree culture medium prior to their 18 hour incubation at 37° C. withaliquots (50 ng) of human leukocyte elastase (HLE), porcine pancreaticelastase (PPE), MMP-2, or Papaine dissolved in the assay buffer (50 mMTris-HCL, pH 7.5 containing 150 mM NaCl, 10 mM CaCl₂, 0.02% Brij and0.02% sodium azide). Each treatment was tested in quadruplicate samples.At the end of the incubation, all samples were microcentrifuged (8000×gfor 5 min) and 100 μl aliquots of supernatant containing the solubilizeddegradation products were mixed with 4 ml scintillation fluid andcounted in triplicates in liquid scintillation counter. Theradioactivity (cpm/sample) released into the supernatant reflecting thedegradation of [³H]-elastin substrate was assessed and the mean andstandard deviations were calculated from sixtiplicate assessments from 3different experiments

Assessment of TA and EA binding to insoluble elastin. In order todirectly show that both polyphenols bind to elastin, triplicate (1 mg)aliquots of our above mentioned preparation of pure insoluble elastinwere incubated with 20 μg/ml of ellagic acid or tannic acid for 2 hr at37° C. The initial concentration of both polyphenols were confirmed by adirect spectrophotometric reading at 280 nm. This method demonstrated adose-dependent linear increase in absorbancy. At the end of incubationperiod the insoluble elastin slurries were separated by centrifugationand the concentrations of polyphenols in supernatants were determinedagain. The detected differences between the initial and finalconcentration of polyphenols in supernatants from particular samplesdirectly indicated that both ellagic acid and tannic acid bound toelastin slurries during the incubation period. In each experimentalgroup means±SD were calculated and obtained values were statisticallycompared with beginning concentrations of both polyphenols.

Statistical analysis. In all above mentioned quantitative assays, meansand standard deviations (expressed as Mean±SD) were calculated andstatistical analyses were carried out by ANOVA to establish whetherdetected differences were statistically significant.

Results.

Ellagic acid and tannic acid enhance deposition of elastin by dermalfibroblasts. Results of immunohistochemical analysis (FIGS. 1 a and b)and quantitative assessment of metabolically labeled insoluble elastin(FIG. 1 c) indicated that 7 day old monolayer cultures of dermalfibroblasts maintained with ellagic acid or tannic acid contain thickerelastic fibers and a higher net content of NaOH-insoluble elastin thanuntreated control cultures. Moreover, results of morphometric analysisdemonstrated that both ellagic acid and tannic acid caused a significant(p<0.005) increase (67±6% and 96±12% respectively) in net elastogenesisobserved in organ cultures of human skin explants maintained for 10 dayswith 5% FBS. Explants maintained for 10 days in culture media containingtannic acid contain thicker and longer elastic fibers than those presentin explants maintained only in control medium or medium with ProK-60(data not shown). Interestingly, the presence of tannic acid seems toparticularly enhance elastogenesis in cells protruding from the stratumbasale, toward the papillary dermis, and in cells surrounding smallcapillaries. Results of semi-quantitative PCR and Northern blottingindicated, however, that treatment of cultured dermal fibroblasts withellagic acid or tannic acid did not induce any increase in thetranscription of their elastin gene (data not shown) nor change theirproliferation rate, as assessed by incorporation of radioactivethymidine and total DNA content (data not shown). Despite this finding,results of western blotting, with anti-tropoelastin antibody, showedthat both cell extracts and conditioned media, of dermal fibroblastsincubated with ellagic acid or tannic acid, contained more intact 70 kDatropoelastin and less immuno-detectable degradation products of lowermolecular weight than untreated counterparts (data not shown). Thisfinding gave evidence that both polyphenols protected newly producedtropoelastin from premature intracellular and pericellular degradationby endogenous proteinases.

Ellagic acid and tannic acid protect elastin from proteolyticdegradation. Results of a pulse and chase experiment (FIGS. 2 a and b)demonstrated that cultures of dermal fibroblasts, exposed for seven daysto ellagic acid and tannic acid, sustain their high net content ofinsoluble elastin (metabolically pulsed with [³H]-valine between day 4and 7) when maintained for an additional seven days (chase period) inmedia containing only 1% FBS (no ellagic acid or tannic acid), which didnot stimulate proliferation (FIG. 2 c) and new elastogenesis. Incontrast, 14 day-old control (untreated) cultures demonstrated asignificant decrease in their net content of metabolically labeledinsoluble elastin (detected at the end of pulse period, at day 7).Moreover, results of an in vitro elastolytic assay demonstrated thatsamples of purified insoluble elastin (purity confirmed by amino acidanalysis), pretreated with ellagic acid or tannic acid, were moreresistant to proteolytic degradation by all tested elastolytic enzymesbelonging to the serine proteinase (human leukocyte elastase and porcinepancreatic elastase), metallo-proteinase (MMP-2) and cysteine proteinase(papaine) families (FIG. 3). These data suggested that ellagic acid andtannic acid protected newly deposited and purified insoluble elastinagainst degradation by both endogenous and exogenous elastolyticenzymes, respectively, through association with elastin.

Ellagic acid and tannic acid bind to elastin and tropoelastin. Resultsof a spectrophotometric assay (displaying a linear concentration curvefor both ellagic acid and tannic acid at an absorbance of 280 nm),comparing concentrations of both polyphenols before and after incubationwith insoluble elastin, demonstrated that 1 mg of pure insolubleelastin, isolated from ligamentum nuchae, absorbed 87±3% of the tannicacid and 81±2% of the ellagic acid in solutions having had initialconcentrations of 20 μg/mL of each polyphenol. This finding implied thatboth polyphenols bind to insoluble elastin. Additional results showedthat preincubation of a [3H]-valine-labeled recombinant peptide,containing the most characteristic hydrophobic and cross-link generatingdomains of tropoelastin, with ellagic acid and tannic acid did notpreclude its effective (practically identical) immunoprecipitation withrespective anti-VGVAPG (SEQ ID NO 19) and anti-AKAAAKAAAKA (SEQ ID NO20) antibodies (data not shown). This indicated that these polyphenolsassociate with and protect tropoelastin in a way which does not obscurehydrophobic domains (eg. VGVAPG (SEQ ID NO 19)), necessary forself-aggregation, nor KAAAK (SEQ ID NO 21) sequences participating incrosslinking.

Ellagic acid and tannic acid enhance elastogenic effect of selectedstimulators of elastogenesis. Results of immunostaining and metaboliclabeling established that addition of ellagic acid or tannic acid tofibroblast cultures simultaneously treated with known stimulators ofelastin gene expression, ProK-60 or Ferric Ammonium Chloride (FAC),significantly enhanced their net deposition of insoluble elastin asestimated in 7 day old monolayer cultures (FIG. 4). Morphometricanalysis additionally demonstrated that both polyphenols significantly(p<0.02) enhanced elastogenic effect of ProK-60 observed in 10 day-oldorgan cultures of human skin explants (Ellagic Acid=22±4% and TannicAcid=35±6%). Representative micrographs depicting synergistic effect ofProK-60 and tannic acid are presented in FIG. 4 b.

Discussion. Results demonstrate that the two polyphenols, ellagic acidand tannic acid, used in concentration of 1 μg/ml, did not modulatecellular proliferation of normal human dermal fibroblasts, despite thefact that anti-proliferative properties of both of these compounds werereported in cultures of various normal and malignant cell lines whenused in higher doses. Dermal fibroblasts treated with both acids did notdemonstrate any increase in levels of elastin mRNA yet facilitated asignificant increase in net elastic fiber content detected byimmunochemistry and metabolic labeling of insoluble elastin. It wasspeculated that both ellagic acid and tannic acid might bind tointracellular tropoelastin and to newly assembled crosslinked elastinand protect them from proteolytic degradation by fibroblast-secretedproteolytic enzymes engaged in early remodeling of extracellular matrix.The hypothesis regarding potential preferential binding of bothpolyphenols to intracellular tropoelastin and extracellular elastinpolymer was based on a previously described observation that addition of0.25% tannic acid to glutaraldehyde fixative dramatically enhancedcontrast of intracellular secretory vesicles containing tropoelastin andcontrast of extracellular elastic fibers in tissues observed under theelectron microscope. In fact, in the pre-immunostaining era, treatmentwith tannic acid became a widely accepted method for ultrastructuralidentification of elastic fibers that were previously described as“electron lucent and amorphous” under electron microscopy. Thehypothesis was further supported by results of our pulse-and-chaseexperiment where ellagic acid and tannic acid pre-treated tropoelastinand insoluble elastin, deposited by dermal fibroblasts, remainedresistant against non-specific endogenous degradation in the absence ofellagic acid or tannic acid in culture medium. Results also show thatpre-incubation of [³H]-labeled pure insoluble elastin, with eitherellagic acid or tannic acid, significantly reduced its rate ofdegradation (in the absence of either polyphenol in the digest buffer)by several exogenous elastolytic enzymes including porcine pancreaticelastase, human leukocyte elastase, papaine and the UVB-inducible MMP-2.Results of this protection assay are consistent with recent observationsthat addition of tannic acid to the glutaraldehyde fixation processincreased the stability of porcine aortic explants exposed to pancreaticelastase digestion. Moreover, the spectrophotometric study demonstratedthat pure insoluble elastin, isolated from ligamentum nuchae, binds bothellagic acid and tannic acid, sequestering them from their solventsolutions. Results of this study are in further agreement theobservation of similar binding of tannic acid by pure aortic elastinover time.

It has been previously shown that plant derived polyphenols, tannins andtheir synthetic derivatives, can inhibit human leukocyte elastase andMMP-2/-9 activity in several tumor cells. Other studies directlydemonstrated that tannic acid specifically inhibits thechymotrypsin-like activity of purified 20S proteasomes and the activityof tissue-type plasminogen activator, urokinase-type plasminogenactivator and plasmin activity. It was speculated that a certainfraction of the described protective effect may be due to a directinhibition of proteolytic enzymes by both polyphenols absorbed by orreleased from the elastin substrate.

The practical biological significance of the observations is thatellagic acid or tannic acid added to cultures of living cells facilitatenormal secretion of tropoelastin and its assembly into elastic fibers byprotecting intra- and extracellular tropoelastin from degradation byunspecific proteinases. Since ellagic acid and tannic acid did not blockdomains responsible for self-aggregation and subsequent cross-linking ofthis protein, it is speculated that ellagic acid and tannic acid may actin concert with the 67-kDa elastin binding protein (EBP) that acts as aprotective molecular chaperone for intracellular tropoelastin. Moreover,the fact that ellagic acid and tannic acid significantly decreaseddegradation of newly produced elastin, in dermal fibroblast cultures,and fully cross-linked elastin, from ligamentum nuchae, indicate thatboth polyphenols may enhance longevity of elastic fibers.

Given the presented beneficial effects of both tested polyphenols, bothmay be used in topical preparations aimed at prevention of elastindegradation characteristic of normal aging and after chronic exposure tosunlight (photoaging). Since ellagic acid and tannic acid did notnegatively interfere with the action of two known stimulators of newelastogenesis, but rather enhanced their net effect, their use incombination with compounds aimed at restoring cutaneous elastic fibersin aged skin or skin of patients afflicted by diseases caused by elastingene insufficiency (i.e. Williams-Beuren Syndrome (WBS) and Cutis Laxa)may also appear beneficial. Results of our recent experiments (data notshown) indicated that tannic acid and ellagic acid prevented rapid,MMP-dependent degradation of tropoelastin produced by dermal fibroblastsderived from three WBS patients, yielding a significant (21-26%) netincrease in deposition of insoluble elastin by these cells. Thus,results of the in vitro studies, presented herein, and lack of clinicalside effects of polyphenolic compounds fully encourage their use in invivo protection of existing elastic fibers and more efficientelastogenesis in skin.

In summary, results of in vitro studies demonstrate the use of tannicacid and ellagic acid and other polyphenolic compounds in skin careproducts aimed at initiation of new elastogenesis and protection ofexisting elastic fibers from normal aging-related and UV-inducedproteolytic degradation.

EXAMPLE 2

In order to directly prove that tannic acid also binds to collagen type1, triplicate (1 mg) aliquots of pure collagen type I were incubatedwith 20 μg/ml of TA for 2 h at 37° C. The initial concentration oftannic acid was confirmed by direct spectrophotometric reading at 280nm. This method adopted from Gori et al. demonstrated a dose-dependentlinear increase in absorbance. At the end of incubation period thecollagen type I slurries were separated by centrifugation and theconcentration of tannic acid in supernatants were spectrophotometricallydetermined again at 280 nm. In each experimental group means±SD werecalculated and obtained values were statistically compared withbeginning concentrations of both polyphenols.

Results. As shown in FIG. 5, the binding studies demonstrate that 1 mgof collagen type I (from rat tail) sequestered 75.5±0.001% (P<0.0001) ofthe tannic acid (originally 20 μg/mL) from solution, suggesting thattannic acid may also bind to collagen type I.

What is claimed is:
 1. A method for reducing photo-induced damage to theskin comprising administering to the skin in need thereof a compositioncomprising about 1 microgram to about 10 micrograms of a polyphenoliccompound and a peptide having a sequence Val-Gly-Val-Ala-Pro-Gly (SEQ IDNO: 19), wherein the photo-induced damage to the skin is reduced.
 2. Themethod of claim 1, wherein said polyphenolic compound is selected fromtannic acid, ellagic acid and combinations thereof.
 3. The method ofclaim 1, wherein the amount of said polyphenolic compound in thecomposition is about 1 μg.
 4. A method of treating damage to the skin ofa subject comprising topically administering to the skin in need thereofa composition comprising about 1 microgram to about 10 micrograms of apolyphenolic compound and a peptide having a sequenceVal-Gly-Val-Ala-Pro-Gly (SEQ ID NO: 19), wherein the deposition ofinsoluble elastin fibers is increased.
 5. The method of claim 4, whereinsaid polyphenolic compound is selected from tannic acid, ellagic acidand combinations thereof.
 6. The method of claim 4, wherein the amountof said polyphenolic compound in the composition is about 1 μg.